Multiple abnormalities have been identified by our group in the serotonergic system in the medulla of SIDS cases. These data are exciting and compelling in light of a large body of experimental data from our group and others that indicate that serotonin (5-HT) neurons are involved in maintaining cardiovascular and respiratory homeostasis and in regulating sleep and arousal. There is strong evidence in particular that 5-HT neurons contribute to the ventilatory and arousal response to hypercapnia, as well as the response to temperature challenges. Thus, a defect in 5-HT neurons fits well with long-standing theories of SIDS proposing that there are defects in cardiorespiratory control, arousal and thermoregulation. Preliminary data from our group also indicate that there are defects in the GABA system in SIDS cases. This is intriguing, because there is a subset of GABA neurons within the raphe nuclei that may also be central chemoreceptors. The work proposed in this project will use in vitro approaches to address cellular and network mechanisms involved in 5-HT and GABA function. We will use state-of-the art in vitro electrophysiological methods to examine the effects of CO2/pH, O2, temperature, gender and nicotine on 5-HT and GABA neurons during development in medullary slices from genetically engineered mice in which all or subsets of serotonin or GABA neurons are fluorescent. With the Neuroanatomy Core, we will also use immunohistochemistry and tract tracing to define the neurochemical organization and connectivity of the medullary 5-HT system relative to the GABA system and the respiratory network. The goal is to use an in vitro approach to provide insight into how a defect in 5-HT and GABA neurons impairs the response of an infant to hypercarbia, hypoxia and/or a temperature challenge, why this defect is expressed only during a critical developmental period, how gender and prenatal exposure to nicotine modifies it, and why death typically occurs during sleep. To accomplish this, we propose the following aims: 1) Define the properties of GABA neurons in the medullary raphe. 2) Characterize how hypoxia, temperature, prenatal nicotine, gender and pH interact to affect different subsets of 5-HT neurons in the medulla at different postnatal ages. 3) Define how network interactions between the raphe, ventrolateral medulla, retrotrapezoid nucleus and pre-B6tzinger Complex influence the response to pH and neuromodulators. The cellular and network experiments proposed here are interdigitated with those in the other Projects of this PPG, and are an intermediate step between the molecular approach of Project 5 and the human and whole animal work of Projects 1-3. Together our results will provide critical insight, we believe, into how a defect in the 5 HT system could lead to SIDS.